1,3,5-Triazine-2,4-diamine, N2-[(1R,2S)-2,3-dihydro-2,6-dimethyl-1H-inden-1-yl]-6-(1-fluoroethyl)-

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Version / remarks:

-draft

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: FIFRA Guideline 72-4 (b)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Controls: yes
- Chemical name of vehicle: Acetone
- Concentration of vehicle in test medium: 0.1 ml/L

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Waterflea
- Source: In-house cultures (Original Culture Source: Aquatic Biosystems, Fort Collins, CO)
- Age of parental stock: First instar < 24 hours old neonates from third brood or older


HOUSING
- Housing conditions:
Subculture Vessels: 400 ml beakers containing approximately 300 ml water
Organism per Vessel: 2
Number of Vessels: 10
System: Static-renewal (approximately 3 water renewals per week)
Water Type: Hard process water (same source as water used for testing)
Feeding Frequency: Daily
Food: Green algae (Pseudokirchneriella subcapitata) and blended TetrafinÒ
flaked fish food
Photoperiod: 16 hours light, 8 hours dark
Light Intensity: 430 – 750 lux
Temperature: 20.0 ± 2.0 °C
Health: Good health, no disease treatments

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 h

Hardness:

174 mg/L as CaCO3

Test temperature:

19.6 to 20.5 °C

pH:

7.6 to 8.6

Dissolved oxygen:

5.5 to 9.2 mg/L (60 to 101 % saturation)

Salinity:

-

Nominal and measured concentrations:

Nominal Test Concentrations: Control, Solvent Control, 0.05, 0.13, 0.32, 0.80, and 2.0 mg a.i./L
Mean Measured Test Concentrations: Control (<0.003), Solvent Control (<0.003), 0.06, 0.14, 0.34, 0.80, and 2.0 mg a.i./L (ppm)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria
- Material, size, headspace, fill volume: Borosilicate Glass; 1 liter beakers containing approximately 900 ml test solution (multiple
organism beakers) and 250 ml beakers containing approximately 200 ml test solution (single organisms beakers)
- Renewal rate of test solution: Static-renewal (approximately 3 water renewals per week)
- No. of organisms per vessel: 5-10
- No. of vessels per concentration (replicates): 3 replicates for sublethal and survival effects assessment with 5 organisms per replicate (multiple organism beakers, reps A, B and C) 10 replicates for sublethal, reproduction and growth effects assessment with one organism per replicate (single organism beakers; reps D to M)
- No. of vessels per control (replicates): 3 replicates for sublethal and survival effects assessment with 5 organisms per replicate (multiple organism beakers, reps A, B and C) 10 replicates for sublethal, reproduction and growth effects assessment with one organism per replicate (single organism beakers; reps D to M)
- No. of vessels per vehicle control (replicates): 3 replicates for sublethal and survival effects assessment with 5 organisms per replicate (multiple organism beakers, reps A, B and C) 10 replicates for sublethal, reproduction and growth effects assessment with one organism per replicate (single organism beakers; reps D to M)


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Intervals of water quality measurement: At experimental start, weekly thereafter including experimental finish

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light, 8 hours dark
- Light intensity: 433 to 740 lux

EFFECT PARAMETERS MEASURED: Sublethal effects, survival (immobilization), time to first brood release, reproduction (neonates per adult reproductive day) and growth (length and dry weight at study termination)
Observation Interval: Observations for sublethal effects and survival made daily, reproductive output (neonates counts) occurred at the time of first brood release and on Monday, Wednesday and Friday there after (to include the day of termination), growth determinations were made at the end of the
exposure.

VEHICLE CONTROL PERFORMED: yes

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.34 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth

Immobilization (Adult):

Percent Immobilized:	Control (7%), Solvent Control (7%), 0.06 (0%), 0.14 (0%), 0.34 (7%), 0.80 (0%) and 2.0 (0%) mg a.i./L
Passed Criteria for Normality and Homogeneity of Variance:	No
Statistical Analysis:	Non-parametric
Treatment Groups Significantly Different (P= 0.05) Controls:	None
No Observed Effect Concentration (NOEC):	2.0 mg a.i./L
Lowest Observed Effect Concentration (LOEC):	>2.0 mg a.i./L
Immobilization Criteria:	Immobilization of parent animals does not exceed 20% at the end of the test as stated in the established validity criteria (USEPA,1996; OECD,1998).

There were no immobilization related differences between the control groups and any of the

treatment groups.

Time to First Brood:

Mean Time to First Brood:	Control (7.4 days), Solvent Control (7.7 days), 0.06 (7.6 days), 0.14 (7.3 days), 0.34 (7.8 days), 0.80 (7.9 days) and 2.0 (8.4 days) mg a.i./L
Culture/Control Brood Development Criteria:	ASTM(2004)suggests that healthy adults will have released a first brood by the time organisms are 7 to 10 days old. This criterion was met for control organisms during this exposure.
Passed Criteria for Normality and Homogeneity of Variance:	No (normality failed)
Statistical Analysis:	Non-parametric
Treatment Groups Significantly Different (P= 0.05) Controls:	2.0 mg a.i./L
No Observed Effect Concentration (NOEC):	0.80 mg a.i./L
Lowest Observed Effect Concentration (LOEC):	2.0 mg a.i./L

Neonates per Adult Reproduction Day:

Mean number of Young/Adult	Control (21.8), Solvent Control (21.1), 0.06 (22.3), 0.14 (21.2), 0.34
Reproductive day:	(21.4), 0.80 (18.4) and 2.0 (7.6) mg a.i./L
Passed Criteria for Normality and Homogeneity of Variance:	No (homogeneity failed)
Statistical Analysis:	Non-parametric
Treatment Groups Significantly Different (P≤ 0.05) Controls:	2.0 mg a.i./L
No Observed Effect Concentration (NOEC):	0.80 mg a.i./L
Lowest Observed Effect Concentration (LOEC):	2.0 mg a.i./L
	Mean number of live offspring produced per parent control animal
Reproductive Criteria:	surviving at the end of the test exceeded the established validity criteria of ≥ 60 (USEPA,1996;OECD,1998).

Growth:

Day of Evaluation:	Day 21 (termination)
Length
Mean Length:	Control (5.23 mm), Solvent Control (5.16 mm), 0.06 (5.29 mm), 0.14 (5.24 mm), 0.34 (5.23 mm), 0.80 (5.09 mm) and 2.0 (4.02 mm) mg a.i./L
Passed Criteria for Normality and Homogeneity of Variance:	Yes
Statistical Analysis:	Parametric
Treatment Groups Significantly Different (P≤ 0.05) Controls:	0.80 and 2.0 mg a.i./L
No Observed Effect Concentration (NOEC):	0.34 mg a.i./L
Lowest Observed Effect Concentration (LOEC):	0.80 mg a.i./L
Dry Weight
	Control (1.465 mg), Solvent Control (1.433 mg), 0.06 (1.443 mg), 0.14
Mean Dry Weight:	(1.417 mg), 0.34 (1.396 mg), 0.80 (1.316 mg) and 2.0 (0.508 mg) mg
	a.i./L
Passed Criteria for Normality and Homogeneity of Variance:	Yes
Statistical Analysis:	Parametric
Treatment Groups Significantly Different (P≤ 0.05) Controls:	0.80 and 2.0 mg a.i./L
No Observed Effect Concentration (NOEC):	0.34 mg a.i./L
Lowest Observed Effect Concentration (LOEC):	0.80 mg a.i./L

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Dec 2012 - 24 Jan 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 850.1350 (Mysid Chronic Toxicity Test)

Version / remarks:

Draft version 1996

Qualifier:

equivalent or similar to guideline

Guideline:

other: ASTM Standard Guide for Conducting Life-Cycle Toxicity Tests with Saltwater Mysids

Version / remarks:

2008

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 0.031, 0.063, 0.13, 0.25 and 0.50 mg/L
- Sampling method: On day 0, 7, 14, 21 and 28 samples were removed from alternating replicate solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Saturator columns were packed with approximately 110 grams of glass wool and attached to the water source. To coat a column, approximately 10 g of the test item was diluted with 300 mL of acetone and poured into the glass column. A vacuum pump was attached and used to draw the solution up and down the column to uniformly coat the wool with the test substance. The column was then attached to a nitrogen gas source to evaporate the remaining acetone. Afterwards, the column was attached to a FLUID Metering, Inc (FMI) pump which delivered a flow of water through the column (48 mL/min or 0.49 L/cycle). For this exposure, a saturator column was prepared prior to exposure initiation, flushed for 7 days before being placed on the system and was made weekly thereafter. Based on measured concentrations, the saturator column delivered a solution of test substance at approximately 2.1 mg/L. FMI pump was calibrated to deliver 0.49 L/cycle of the 2.1 mg/L saturator column solution to the diluter system's mixing chamber. The mixing chamber was positioned over a magnetic stir plate which aided in the mixing of the stock solution. The solution contained in the mixing chamber constituted the highest nominal test concentration and was subsequently diluted (50%) to provide the remaining nominal exposure concentrations.
- Differential loading: no
- Controls: yes, dilution water

Test organisms (species):

Americamysis bahia (previous name: Mysidopsis bahia)

Details on test organisms:

TEST ORGANISM
- Common name: mysid
- Source: cultures maintained at Smithers Viscient, Wareham, USA, originally obtained from MBL Aquaculture located in Sarasota, USA
- Age of parental stock: maintained in house for approximately 12 months prior to use
- Feeding during test: yes (live brine shrimp (Artemia salina) ≤ 48 hours old (post-hydration), twice daily)
- Breeding conditions: recirculating 80 L glass aquaria containing dilute, natural seawater (salinity range: 21 - 22‰, dissolved oxygen: 83.6 - 92.4%, pH: 7.7, temperature 24°C - 26°C, photoperiod: 16 h light and 8 h dark). Undergravel filters were used to provide aeration and a current conducive to feeding.

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

28 d

Test temperature:

25°C - 27°C

pH:

7.2 - 8.0

Dissolved oxygen:

4.74 - 7.25 mg/L

Salinity:

19 - 21‰

Nominal and measured concentrations:

Nominal concentrations: 0, 0.031, 0.063, 0.13, 0.25 and 0.50 mg/L
Mean measured concentrations: < LOQ, 0.040, 0.069, 0.12, 0.25 and 0.50 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass test aquaria (30 x 15 x 20 cm, 4.5 L), for the first 12 days the aquaria contained retention chambers: glass petri dishes (10 cm diameter, fill volume: 785 mL), from day 13 the aquaria contained pairing chambers: petri dishes (diameter: 6 cm, fill volume: 250 mL)
- Type of flow-through: intermittent-flow proportional diluter
- Renewal rate of test solution (frequency/flow rate): 7.7 aquarium volume additions per day (90% test solution replacement rate:approximately 7 hours)
- No. of organisms per vessel: 20 (F0 Generation), 10 (F1 Generation)
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- F0 Life-Cycle Exposure Initiation: Each group of 20 mysids was impartially transferred to the retention chambers. The exposure was initiated when the retention chambers were placed in their respective exposure aquaria. Each exposure aquarium contained one retention chamber, yielding 20 mysids per replicate vessel and 80 organisms for each treatment level and the control.
- F0 Life-Cycle Mysid Pairing: When sexual maturity was reached (day 13) one mature male and one mature female were randomly assigned to each of the pairing chambers (with a maximum of five male/female pairs per replicate). Unpaired mysids were pooled and maintained in one of the initial retention chambers until they were paired or until test termination. When three or fewer unpaired mysids remained in a retention chamber, they were transferred to a separate pairing chamber. Male mysids from this pool were used to replace dead males from the paired (male/female) groups. Females that died in the pairing chambers were replaced at the Study Director’s discretion. For example, if a female within a pairing chamber died prior to initiation of reproduction, that female may be replaced. Once reproduction within the study had begun, females were not replaced.
F1 Generation: groups of 10 offspring replicate, 40 per treatment (if 10 offspring could not be collected, a reduced number of young was collected and evaluated; n > 5) were removed from mysid chambers in each replicate vessel and placed in separate pairing chambers in that replicate.
Feeding: live brine shrimp (Artemia salina) nauplii, ≤ 48 hours old twice a day, at least one of these feedings was with brine shrimp nauplii enriched with Selco®, a substance high in saturated fatty acids. F0 retention chambers :90 nauplii/mysid (test days 0 - 3), 135 nauplii/mysid (test days 4 - 6), 180 nauplii/mysid (test days 7 - 9), 225 nauplii/mysid (test day 10 - pairing). From dayof pairing until test termination: 450 nauplii/mysid, F1 chambers: 90 nauplii/mysid

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilute, filtered, natural seawater from the Cape Cod Canal, Bourne, USA collected from about 1 to 4 meters offshore at a depth of approximately 0.5 meters. The seawater was then transferred by a pump (fiberglass reinforced thermoplastic housing) through polyvinyl chloride (PVC) pipes and transported to the laboratory in a 3400 L fiberglass holding tank. In the laboratory, the seawater was adjusted to a salinity of 20 ± 3‰ with laboratory well water, filtered through 20-, 5 µm and 1 µm polypropylene core filters prior to use. The seawater was pumped under constant pressure through PVC pipes to the intermittent-flow proportional diluter system.
- Total organic carbon: 0.96 - 1.2 mg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature, dissolved oxygen concentration, pH and salinity were measured in each replicate (day 0), alternating replicates of treatment groups and control (daily).

OTHER TEST CONDITIONS
- Photoperiod: 16 h light and 8 h dark
- Light intensity: 880 - 1100 lux

EFFECT PARAMETERS MEASURED:
- length of time for brood appearance (i.e., gravid females) was noted for all first generation mysids prior to pairing
- numbers of dead and living organisms
- abnormal appearance or behavior
- number of offspring produced by each individual female
- body length measurements
- dry body weight

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately 2
- Preliminary test: yes
- Test concentrations: 0, 0.013, 0.025, 0.050, 0.10, 0.20 mg/L
- Results used to determine the conditions for the definitive study: F1 generation 96-hour post-release survival among mysids exposed to the 0.025, 0.050 and 0.10 mg/L treatment levels averaged 95%. F1 generation 96-hour post-release survival in the control and the remaining treatment levels tested (0.013 and 0.20 mg/L) averaged 100%.

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

0.25 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.25 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Male body length, male and female body weight

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

0.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Male body length, male and female body weight

Details on results:

- no effect on survival in any treatment group

Reported statistics and error estimates:

All statistical conclusions were made at the 95% level of certainty except in the case of the basic assumption tests, e.g., Shapiro-Wilks’ Test (U.S. EPA, 2002) and Bartlett’s Test (U.S. EPA, 2002), in which the 99% level of certainty was applied. The assumption that observations are normally distributed and homogeneous must be validated before parametric procedures can be performed. If the data are not normally distributed, then a non-parametric procedure is used for subsequent analyses. All endpoints were assessed in this fashion with the exception of survival endpoints (e.g., 28-day survival, male and female survival and F1 survival). Since the survival endpoints are binominal data, they were analyzed using a Model III 2 x 2 contingency table. The following procedures were used:
- Data for the survival endpoints (e.g., 28-day survival, male and female survival and F1 survival) were analyzed using Fisher’s Exact Test with Bonferroni-Holm’s Adjustment (U.S. EPA, 2002).
- Shapiro-Wilk’s Test for normality (U.S. EPA, 2002) was conducted and compared the observed sample distribution with a normal distribution. For this study, all data met this assumption.
- As a check on the assumption of homogeneity of variance, data for each endpoint were analyzed using Bartlett’s Test (U.S. EPA, 2002). For this study, all data met this assumption.
- All endpoints met the assumptions of normal distribution and homogeneity; therefore, Dunnett's Multiple Comparison Test (U.S. EPA, 2002), a parametric procedure, was used to evaluate the data.

Table 1: Summary of the first generation (F0) reproductivesuccess (offspring per female) at termination of the 28-day life-cycle exposure of mysids (Americamysis bahia)

to BCS-AA10717.

Mean Measured Concentration(mg/L)   Percent of Females Producing Young Number of Offspring Per Female Control A 100 24.8   B 80 13.6   C 100 14.8   D 100 19.0   Mean (SD)a 95 (10) 18.1 (5.1) 0.040 A 80 11.0   B 100 17.8   C 80 12.4   D 80 9.6   Mean (SD) 85 (10) 12.7 (3.6) 0.069 A 80 10.8   B 100 13.6   C 100 8.4   D 100 19.2   Mean (SD) 95 (10) 13.0 (4.6) 0.12 A 100 12.4   B 100 17.0   C 100 17.2   D 100 15.4   Mean (SD) 100 (0) 15.5 (2.2) 0.25 A 80 13.0   B 100 8.8   C 60 10.6   D 100 11.6   Mean (SD) 85 (19) 11.0 (1.8)b 0.50 A 80 9.2   B 60 4.2   C 40 3.2   D 60 5.4   Mean (SD) 60 (16) 5.5 (2.6)b

a     Mean values are presented with standard deviations (SD) in parentheses.

b    Significantly reduced compared to the control, based on Dunnett’s Multiple Comparison Test.

Table 2: Summary of mean total body length measurements of firstgeneration (F0) male and female mysids measured at thetermination of the 28-day life-cycle test exposing mysids (Americamysis bahia) to BCS-AA10717.

 

Mean Measured Concentration (mg/L)		Mean Total Body Length (mm)
		Males	Females
Control	A	7.03	7.10
	B	7.14	7.30
	C	7.00	7.59
	D	7.06	7.20
	Mean (SD)a	7.06 (0.06)	7.30 (0.21)
0.040	A	6.83	6.90
	B	6.62	7.26
	C	6.90	7.69
	D	7.08	7.01
	Mean (SD)	6.85 (0.19)	7.21 (0.35)
0.069	A	7.11	7.31
	B	7.15	7.41
	C	7.41	7.37
	D	7.21	7.41
	Mean (SD)	7.22 (0.14)	7.37 (0.05)
0.12	A	6.97	7.27
	B	7.29	7.43
	C	7.15	7.24
	D	7.08	7.37
	Mean (SD)	7.12 (0.13)	7.33 (0.09)
0.25	A	6.92	7.08
	B	7.16	7.35
	C	7.16	7.55
	D	6.83	7.13
	Mean (SD)	7.02 (0.17)	7.28 (0.22)
0.50	A	6.32	7.09
	B	6.85	7.14
	C	6.29	6.84
	D	6.61	6.98
	Mean (SD)	6.52 (0.26)b	7.01 (0.13)

a     Mean values are presented with standard deviations (SD) in parentheses.

b    Significantly reduced compared to the control, based on Dunnett’s Multiple Comparison Test.

Table 3: Summary of dry body weight measurements of firstgeneration (F0) male and female mysids measured at the termination of the 28-day life-cycle test exposing mysids (Americamysis bahia) to

BCS-AA10717.

Mean Measured Concentration (mg/L)   Dry Body Weight (mg)   Males Females Control A 0.93 0.99   B 0.89 1.25   C 0.82 1.29   D 0.83 1.26   Mean (SD)a 0.87 (0.05) 1.20 (0.14) 0.040 A 0.78 1.09   B 0.89 1.06   C 0.89 1.40   D 0.88 1.03   Mean (SD) 0.86 (0.05) 1.14 (0.17) 0.069 A 0.90 1.05   B 0.86 0.96   C 0.94 1.26   D 0.85 1.21   Mean (SD) 0.89 (0.04) 1.12 (0.14) 0.12 A 0.86 1.08   B 0.85 1.30   C 0.90 1.09   D 0.79 1.05   Mean (SD) 0.85 (0.05) 1.13 (0.11) 0.25 A 0.82 1.02   B 0.86 1.10   C 0.79 1.23   D 0.75 1.08   Mean (SD) 0.80 (0.05) 1.11 (0.09) 0.50 A 0.64 0.88   B 0.73 0.97   C 0.68 0.83   D 0.67 0.88   Mean (SD) 0.68 (0.04)b 0.89 (0.06)b

a     Mean values are presented with standard deviations (SD) in parentheses.

b    Significantly reduced compared to the control, based on Dunnett’s Multiple Comparison Test.

Analytical Results:

Table 4: Concentrations of BCS-AA10717 measured in exposure solutions during the28-daylife-cycle exposure of mysids (Americamysis bahia).

Nominal Concentration (mg/L)                                      Measured Concentration(mg/L)                                         Mean (SD)a Percent Nominala(%)   Range of Mean Measured 1.5 Variability Day 0 Day 7 Day 14 Day 21 Day 28 % CV Control < 0.0027b < 0.0024 < 0.0025 < 0.0025 < 0.0026 NAc (NA) NA NA NA NA 0.031 0.048 0.039 0.037 0.032 0.044 0.040 (0.0062) 130 16 80 - 120 1.5 0.063 0.087 0.062 0.065 0.054 0.076 0.069 (0.013) 110 18 79 - 130 1.6 0.13 0.15 0.12 0.12 0.095 0.13 0.12 (0.020) 96 16 76 - 120 1.6 0.25 0.31 0.23 0.23 0.18 0.30 0.25 (0.053) 100 22 73 - 120 1.7 0.50 0.62 0.47 0.47 0.38 0.54 0.50 (0.090) 99 18 76 - 130 1.6 QCd#1 0.0161 0.0165 0.0170 0.0160 0.0163           0.0156 (103) (106) (109) (102) (104)           QC #2 0.135 0.127 0.132 0.121 0.127           0.125 (108) (102) (106) (96.6) (102)           QC #3 0.555 0.557 0.526 0.473 0.500           0.500 (111) (111) (105) (94.6) (99.9)

a         Mean measured concentrations, standard deviations (SD) and percent of nominal were calculated using actual analytical data and not the rounded (2 significant figures) data presented in this table.

b Concentrations expressed as less than values were below the limit of quantitation (LOQ). The LOQ for each analysis is dependent upon the regression, the area of the low standards and the dilution factor of the controls.

c         NA = NotApplicable.

d     QC = Quality Control sample. The percent recovery for the corresponding QC sample is presented in parentheses.

Table 5: Acceptability criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
Less than 30% of the first generation control mysids die between pairing and the end of the test.	17%	yes
More than 25% of the paired first generation females in the controls produce young.	95%	yes
The average number of young produced by the paired first generation females in the control(s) is more than 3 offspring per female.	18.1	yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

NOEC freshwater (21 d): 0.34 mg a.s./L (measured, OECD 211, Daphnia magna, reproduction)

NOEC saltwater (28 d): 0.12 mg a.s./L (measured, EPA OPPTS 850.1350, Americamysis bahia, reproduction)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.34 mg/L

Marine water invertebrates

Marine water invertebrates

Effect concentration:

0.12 mg/L

Additional information

Two experimental GLP studies are available testing the chronic toxicity of indaziflam towards aquatic invertebrates. One study was performed with water flea Daphnia magna and one study was performed with saltwater mysid Americamysis bahia.

In the key freshwater study according to OECD 202, D. magna was exposed to nominal concentrations ranging from 0.05 to 2.0 mg a.s./L, a solvent control (0.1 mL acetone/L) and a dilution water control.Mean measured test concentrations ranged from approximately 100% to 113% of nominal, indicating that the nominal concentrations were achieved. The NOEC for reproduction after 21 days was determined to be 0.34 mg a.s./L (mean measured).

In the marine toxicity study according to a draft version of EPA OPPTS 850, saltwater mysids (A. bahia) were exposed for 28 days in a flow-through system to six concentrations of the test substance (nominal concentrations: 0.031 - 0.50 mg a.s./L) and a dilution water control. Mean measured test concentrations ranged from approximately 96% to 130% of nominal. The results of the study were based on the mean measured concentrations. The NOEC (28 d) for mysid reproduction was 0.12 mg a.s./L.